Pelvic organ tumors in men and women are estimated to account for 48% and 21%, respectively, of new tumors diagnosed in the United States in 2012 according to the American Cancer Society (Cancer Facts & Figures, 2012). While irradiation is a key therapy for treating these malignancies, the dose is limited by the potential for developing radiation cystitis. The acute phase of cystitis includes inflammation and bladder overactivity resulting from disruption of the urothelial cell (UC) permeability barrier and sensitization of afferent nerves. A chronic phase can develop in 6-12 months with vascular endothelial cell damage, ischemia, collagen deposition and decreased bladder compliance. During the first grant period, we used nitric oxide (NO?), nitrite (NO2-) and peroxynitrite (ONO2-) microsensors along with Ca2+ imaging to determine that irradiation induces Ca2+ influx and release in UC that activate NO? synthase (NOS). NO? binds to cytochrome oxidase, resulting in superoxide (*O2-) and ONO2- which inhibits mitochondrial respiration. This leads to swelling and rupture of the mitochondria, cytochrome c release and apoptosis. We developed a mitochondrial targeting strategy (U.S. Patent 11/565,779) for NOS antagonists and free radical scavengers, and mouse models where the bladder or descending colon is withdrawn through a small incision and selectively irradiated. These agents were radioprotective when intravesically instilled prior to bladder, but not colonic, irradiation (10 Gy; 1 Gy=100 rads). On the other hand, intracolonic administration of the antagonists blocked cross-sensitization and cystitis due to colonic irradiation. This suggests that pelvic organ cross-sensitization is involved in the development of radiation cystitis and that agents must be given systemically to afford multiple organ protection. In this proposal, we will investigate the mechanism for pelvic organ cross-sensitization, the channels involved in Ca2+ influx and release responsible for NOS activation and the benefits of new therapeutic agents to prevent or treat radiation cystitis. We have three specific aims that will be tested in control and transient receptor potential ankyrin 1 and vanilloid 1 (TRPA1-/- and TRPV1-/-) knockout mice using optical mapping of UC, dorsal root ganglia (DRG) neurons and afferent nerve terminals in the bladder wall and spinal cord. Cells and tissues will be stained with voltage- or Ca2+-sensitive dyes and/or afferent nerves labeled using pseudorabies virus (PRV369 and PRV823) expressing the genetically encoded Ca2+ indicator, GCaMP4. TRPA1-/- and TRPV1-/- mice will be used to determine the roles of these channels in irradiation-induced damage and HPLC-MS will be used to identify irradiation-induced electrophilic fatty acid derivatives (EFAD) involved in TRPA1 and TRPV1 channel activation. The therapeutic benefits of nitro-fatty acids (FA-NO2) and brain-derived neurotrophic factor- and nerve growth factor-antibodies (BDNF-AB and NGF-AB) will be tested. Our three specific aims and associated hypotheses are: 1 Determine the mechanism of bladder-colon afferent cross-sensitization that contributes to radiation cystitis. Hypothesis: Cystitis can develop from colonic irradiation due to cross-sensitization of divergent afferent nerves in the periphery or convergent ones in the spinal cord. 2 Determine the roles of TRPA1 and TRPV1 in irradiation-induced urothelial damage and afferent sensitization. Hypothesis: Irradiation produces EFAD that activate TRPA1 and TRPV1 ion channels resulting in UC damage and afferent sensitization. 3 Determine the therapeutic benefits of new agents for the prevention or treatment of radiation cystitis. Hypothesis: FA-NO2 are radioprotective by desensitizing TRPA1 and TRPV1 channels and BDNF-AB and NGF-AB are therapeutic by inhibiting nerve growth and sensitization in bladder and spinal cord.